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spinqualitylabelsall models 1ghd, resolution 2.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Crystal structure of the glutaryl-7-aminocephalosporanic acid acylase by mad phasing

Overview

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130), was irreversibly inhibited in a time-dependent manner by two substrate, analogs bearing side chains of variable length, namely 7beta-bromoacetyl, aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl, aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with, BA-7-ACA was attributable to reaction with a single amino acid residue, within the beta-subunit proven by comparative matrix assisted laser, desorption/ionization-time of flight mass spectrometry. Further mass, spectrometric analysis demonstrated that the fourth tryptophan residue of, the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC, spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that, tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon, bond. Replacing Trp-B4 with other amino acid residues caused increases in, K(m), decreases in k(cat), and instability of enzyme activity. None of the, mutant enzymes except W-B4Y could be affinity-alkylated, but all were, competitively inhibited by BA-7-ACA. Kinetic studies revealed that both, BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well, as Tyr-B4 of the mutant W-B4Y. Because these alkylations were, energy-requiring under physiological conditions, it is likely that the, affinity labeling reactions were catalyzed by the C130 enzyme itself. The, Trp-B4 residue is located in the middle of a characteristic, alphabetabetaalpha sandwich structure. Therefore, a large conformational, alteration during inhibitor binding and transition state formation is, likely and suggests that a major conformational change is induced by, substrate binding during the course of catalysis.

About this Structure

1GHD is a Protein complex structure of sequences from Pseudomonas sp. 130. Full crystallographic information is available from OCA.

Reference

Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130., Huang X, Zeng R, Ding X, Mao X, Ding Y, Rao Z, Xie Y, Jiang W, Zhao G, J Biol Chem. 2002 Mar 22;277(12):10256-64. Epub 2002 Jan 8. PMID:11782466

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